1. Field of the Invention
The present invention relates generally to the field of molecular biology. More specifically, the invention relates to plant isoflavonoid hydroxylase genes and methods of use thereof.
2. Description of the Related Art
Isoflavonoids are a subclass of phenylpropanoid metabolites distributed primarily in legumes (Dixon and Sumner, 2003). They possess a wide range of biological activities (Dixon, 1999), but most research has focused on their functions as pathogen-inducible antimicrobial compounds (phytoalexins) (Ingham, 1982; Dewick, 1993; Dixon, 1999) or as dietary phytoestrogens implicated in human disease prevention (Adlercreutz and Mazur, 1997; Dixon and Ferreira, 2002). Different legume species produce different classes of isoflavonoid phytoalexins, of which substituted pterocarpans, such as medicarpin from alfalfa and pisatin from pea, are the best known.
Complex isoflavonoid derivatives such as the rotenoids rotenone, deguelin, and amorphigenin from Amorpha, Lonchocarpus, Derris, and Tephrosia species possess insecticidal and parasiticidal properties (Lambert et al., 1993; Nicholas et al., 1985). Maackiain, which accumulates along with medicarpin (the major phytoalexin in Medicago species) in red clover (Trifolium pratense), subterranean clover (T. subterraneum), and chickpea (Cicer arietinum) (Dewick and Ward, 1978; Higgins, 1972; Ingham, 1982), has recently been shown to have larvicidal activity against caterpillars of Heliocoverpa armigera that attack chickpea (Simmonds and Stevenson, 2001).
The biosynthesis of complex isoflavonoids such as the antimicrobial pterocarpans requires hydroxylation of the isoflavonoid nucleus at either the 2′ and/or 3′ positions. Isoflavone 2′-hydroxylase (I2′H) activity has been identified in microsomal fractions of elicited cells of soybean (Kochs and Grisebach, 1986), chickpea (Clemens et al., 1993; Gunia et al., 1991; Hinderer et al., 1987) and alfalfa (Medicago sativa) (Choudhary et al., 1990), and an I2′H (CYP81E1) gene characterized from licorice (Glycyrrhiza echinata L). Recombinant CYP81E1 catalyzed the 2′-hydroxylation of formononetin (7-hydroxy, 4′-methoxyisoflavone) and the 2′- and 3′-hydroxylation of daidzein (7, 4′-dihydroxyisoflavone) in vitro in yeast microsomes (Akashi et al., 1998). Several cDNA clones with high sequence identity to I2′H have been isolated from elicited Lotus japonicus and chickpea cell suspension cultures by PCR strategies based on P450 conserved motifs (Overkamp et al., 2000; Shimada et al., 2000). However, functional characterization has not been reported.
Hydroxylation at the 3′-position of the B-ring of an isoflavone is a key step in the formation of the methylenedioxy bridge of maackiain (Clemens and Barz, 1996; Clemens et al., 1993; Dewick and Ward, 1978), and in the formation of rotenoids (Dixon, 1999). Isoflavone 3′-hydroxylase (I3′H) activities have been detected in the fungus Fusarium (Mackenbrock and Barz, 1983); in roots, leaves and elicited cell suspension cultures of chickpea (Clemens et al., 1993; Hinderer et al., 1987); and more recently in human liver (Tolleson et al., 2002) in which P450 enzymes are presumably involved in isoflavone catabolism.
While the foregoing studies have provided a further understanding of the metabolism of plant secondary metabolism, genes encoding I3′H have not yet been identified. Further, functional characterization of genes encoding I2′H has not been carried out in plants. The identification and characterization of such genes encoding isoflavone hydroxylases would allow the creation of novel plants with improved phenotypes and methods for use thereof. There is, therefore, a great need in the art for the identification of plant isoflavonoid hydroxylase genes.